Viruses of the Parvoviridae family are small DNA animal viruses characterized by their ability to infect particular hosts, among other factors. Specifically, the family Parvoviridae is divided between two subfamilies: the Parvovirinae, which infect vertebrates, and the Densovirinae, which infect insects. The subfamily Parvovirinae (members of which herein are referred to as the parvoviruses) includes the genus Dependovirus, the members of which genus are unique in that, under most conditions, these viruses require coinfection with a helper virus such as adenovirus or herpes virus for productive infection in cell culture. The genus Dependovirus includes adeno-associated virus (AAV), which normally infects humans (e.g., serotypes 2, 3A, 3B, 5, and 6) or primates (e.g., serotypes 1 and 4), and related viruses that infect other warm-blooded animals (e.g., bovine, canine, equine, and ovine adeno-associated viruses). The parvoviruses and other members of the Parvoviridae family are generally described in Kenneth I. Berns, “Parvoviridae: The Viruses and Their Replication,” Chapter 69 in FIELDS VIROLOGY (3d Ed. 1996).
In recent years, AAV has emerged as a preferred viral vector for gene therapy due to its ability to efficiently infect both nondividing and dividing cells, integrate into a single chromosomal site in the human genome, and pose relatively low pathogenic risk to humans. In view of these advantages, recombinant adeno-associated virus (rAAV) presently is being used in gene therapy clinical trials for hemophilia B, malignant melanoma, cystic fibrosis, and other diseases.
AAV is able to infect a number of mammalian cells. See, e.g., Tratschin et al., Mol. Cell Biol., 5(11):3251-3260 (1985) and Grimm et al., Hum. Gene Ther., 10(15):2445-2450 (1999). However, AAV transduction of human synovial fibroblasts is significantly more efficient than in similar murine cells, Jennings et al., Arthritis Res, 3:1 (2001), and the cellular tropicity of AAV significantly differs between serotypes. See, e.g., Davidson et al., Proc. Natl. Acad. Sci. USA, 97(7):3428-3432 (2000) (discussing differences among AAV2, AAV4, and AAV5 with respect to mammalian CNS cell tropism and transduction efficiency). Most commonly, rAAV is produced in 293 cells, COS cells, HeLa cells, KB cells, and other mammalian cell lines. See, e.g., U.S. Pat. Nos. 6,156,303, 5,387,484, 5,741,683, 5,691,176, and 5,688,676; U.S. Patent Application 2002/0081721, and International Patent Applications WO 00/47757, WO 00/24916, and WO 96/17947. Although virus-like particles (VLPs) of parvoviruses have been produced in insect cells (see, e.g., Ruffing et al., J. Virol., 66(12):6922-6930 (1992), Brown et al., J. Virol., 65(5):2702-2706 (1991), and Yuan et al., Virology, 279(2):546-547 (2001)), the production of infectious AAV in nonmammalian, invertebrate cells currently is not known. The replication of parvoviral viral genomes, including, particularly, Dependovirus genomes, in nonmammalian, invertebrate cells, is similarly heretofore unknown.
The difficulties involved in scaling-up rAAV production for clinical trials and commercialization using current mammalian cell production systems can be significant, if not entirely prohibitive. For example, for certain clinical studies more than 1015 particles of rAAV may be required. To produce this number of rAAV particles, transfection and culture with approximately 1011 cultured human 293 cells, the equivalent of 5,000 175-cm2 flasks of cells, would be required. Related difficulties associated with the production of AAV using known mammalian cell lines are recognized in the art. See, e.g., Grimm et al, supra. There also is the possibility that a vector destined for clinical use produced in a mammalian cell culture will be contaminated with undesirable, perhaps pathogenic, material present in a mammalian cell.
In view of these and other issues there remains a need for alternative and improved methods of efficiently, safely, and economically producing a large amount of infectious rAAV particles. The invention provides such methods. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.